ABA signaling converts stem cell fate by substantiating a tradeoff between cell polarity, growth and cell cycle progression and abiotic stress responses in the moss Physcomitrium patens.

Abscisic acid (ABA)-mediated abiotic stress tolerance causes plant growth inhibition. Under such stress conditions, some mosses generate de novo stress-resistant stem cells, also called brood cells or brachycytes, that do not exist under normal conditions. However, the cell physiological basis of the growth inhibition and the stem cell formation is not well understood. Here, we show that the ABA-induced growth inhibition of the moss Physcomitrium patens apical protonemal cells (protonemal stem cells) is mediated through a shift from asymmetric to symmetric cell division. This change of the cell division mode, and consequently change of stem cell activity, is substantiated by dampening cell polarity and cell proliferative activity through the altered distribution of cytoskeletal elements, the mitotic spindle and the vacuole, which results in the production of stress-resistant stem cells. Alteration of the cell physiological data is supported by the results of RNAseq analysis indicating rapid changes in both cell polarity and cell cycle regulation, while long-term treatments with ABA for 5 to 10 days impact mainly the transcriptional and translational regulation. The regulation of cell polarity and cell cycle genes suggests growth arrest mediated by small GTPases (ROPs) and their guanine exchange factors (ROPGEFs) and by cyclin and cyclin-dependent-kinase complex, respectively. Our data suggest that a tradeoff relationship between growth ability and abiotic stress response in the moss is substantiated by ABA signaling to suppress cell polarity and asymmetric cell growth and may play a pivotal role in stem cell fate conversion to newly produced stress-resistant stem cells.

Castanospermine suppresses CD44 ectodomain cleavage as revealed by transmembrane bioluminescent sensors.

Cluster of differentiation 44 (CD44) is a single-pass transmembrane glycoprotein that is a widely distributed cell-surface adhesion molecule. CD44 undergoes ectodomain cleavage by membrane-associated metalloproteinases in breast cancer cells. Cleavage plays a critical role in cancer cell migration by mediating the interaction between CD44 and the extracellular matrix. To explore inhibitors of CD44 ectodomain cleavage, we developed two bioluminescent sensors for the detection of CD44 ectodomain cleavage. The sensors were designed as two-transmembrane proteins with split-luciferase fragments, one of which was cyclized by protein trans-splicing of a DnaE intein. These two sensors emit light by the cyclization or the spontaneous complementation of the luciferase fragments. The luminescence intensities decreased upon cleavage of the ectodomain in breast cancer cells. The sensors revealed that castanospermine, an α-glucosidase inhibitor, suppressed the ectodomain cleavage of endogenous CD44 in breast cancer cells. Castanospermine also inhibited breast cancer cell invasion. Thus, the sensors are beneficial tools for evaluating the effects of different inhibitors.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

[Efforts toward Venous Access of Anti-Cancer Drug by Nurses].

Our hospital has an established outpatient chemotherapy room, and medical doctors have accessed veins for infusion so far. We trialed venous access by nurses for the purpose of managing safe and proper cancer chemotherapy, reducing the work of doctors, and reducing patient waiting time. A questionnaire was conducted in June 2013, and nurses secured routes at 19 facilities(58%)of the 33 national university hospitals. In November of the same year, the working group was established, and from September 2016 to March 2017, lectures, practical skills, a paper test, and a practical test were conducted; successful applicants were approved as in-hospital certified nurses. From April 2017, we started intravenous injection of anti-cancer drugs by nurses in outpatient chemotherapy rooms and always waiting for doctor in chemotherapy room. There have been many favorable reports of reduced pain and less route failure from patients, and issues, such as extravasation and dyspnea, have not occurred yet. The doctors who were interrupting their work by 29 minutes(20 minutes to and from the patient for a 9-minute procedure)could concentrate on their own tasks. However, patient waiting time increased from 14 minutes to 21 minutes because the amount of work for nurses increased. In the future, reducing the burden on nurses, for example, by increasing the number of nurses, is warranted.

Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.

Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor needs to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from the cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments.

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase-fragment complementation.

Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-µ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor.

Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity.

BACKGROUND: Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. METHODS: We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. RESULTS: The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. CONCLUSION: By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions.

p62/SQSTM1 plays a protective role in oxidative injury of steatotic liver in a mouse hepatectomy model.

AIMS: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. RESULTS: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. INNOVATION: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. CONCLUSION: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt, caused posthepatectomy necrotic/apoptotic liver injury and delayed regeneration, both mainly via a redox-dependent mechanism.

Hes1 is required for contact inhibition of cell proliferation in 3T3-L1 preadipocytes.

Cell-cell contact causes the growth arrest of cells in culture, which is referred to as contact inhibition of cell proliferation. Notch signaling is involved in the growth arrest of cells represented by contact inhibition of cell proliferation. The Notch effector, Hes1 (Hairy and enhancer of split 1), promotes or inhibits cell proliferation by repressing the expression of cyclin-dependent kinase inhibitors. However, it is still unclear whether Hes1 is involved in the mechanisms responsible for contact inhibition of cell proliferation. Here, we examined the involvement of Hes1 in contact inhibition of cell proliferation using a γ-secretase inhibitor and a stable 3T3-L1 preadipocyte cell line expressing Hes1-shRNA as a model cell. The cell cycle was not arrested in Hes1-knockdown cells even after the cells reached confluence. Reduced Hes1 levels failed to repress the expression of E2F-1, a transcription factor required for the progression of the cell cycle. The expression of Myc, cyclin E1, and cyclin A2 in E2F-1 target genes was also higher in Hes1-knockdown cells compared with the negative control. These results suggest that Hes1 plays essential roles in contact inhibition of cell proliferation in 3T3-L1 cells by repressing E2F-1 expression.